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The present invention relates a 3-dimensional cell to tissue and maintenance process, more particularly to methods of culturing cells in a culture environment, either in space or in a gravity field, with minimum fluid shear stress, freedom for 3-dimensional spatial orientation of the suspended particles and localization of particles with differing or similar sedimentation properties in a similar spatial region.
This research addressed a number of issues relevant to the performance of teams in demanding environments. Initial work, conducted in the aviation analog environment, focused on developing new measures of performance related attitudes and behaviors. The attitude measures were used to assess acceptance of concepts related to effective teamwork and personal capabilities under stress. The behavioral measures were used to evaluate the effectiveness of flight crews operating in commercial aviation. Assessment of team issues in aviation led further to the evaluation and development of training to enhance team performance. Much of the work addressed evaluation of the effectiveness of such training, which has become known as Crew Resource Management (CRM). A second line of investigation was into personality characteristics that predict performance in challenging environments such as aviation and space. A third line of investigation of team performance grew out of the study of flight crews in different organizations. This led to the development of a theoretical model of crew performance that included not only individual attributes such as personality and ability, but also organizational and national culture. A final line of investigation involved beginning to assess whether the methodologies and measures developed for the aviation analog could be applied to another domain -- the performance of medical teams working in the operating room.
The ultimate source of nutrients for all living organisms consists of the inanimate nutrient reserves found on earth. Of the elements known to exist, seven are considered essential to plants in large amounts (macronutrients), and many others are required in smaller quantities (micronutrients). Essentiality of a nutrient is defined according to the following concepts: (a) A deficiency of the element makes it impossible for the plant to complete the vegetative or reproductive stage of its cycle; (b) such deficiency is specific to the element in question and can be prevented or corrected only by supplying this element; (c) the element is directly involved in the nutrition of the plant quite apart from its possible effects in correcting some unfavorable microbiological or chemical condition of the soil or other culture medium. From that standpoint a favorable response from adding a given element to the culture medium does not constitute conclusive evidence of its indispensability in plant nutrition. All the elements occurring in the outer part of the earth are in constant turnover among the different components of earth. This overall migration is referred to as geochemical cycling. When cycling includes a role for biological organisms, it is referred to as "biogeochemical cycling." Like most cyclical processes in nature, the biogeochemical cycling of elements is not continuous, nor does it proceed in a well-defined direction. At stages, it may be halted or short-circuited, or it may change. Any changes will eventually impact the survival, evolution, and development of biological species in the system. The relationship of the various systems is represented in a schematic manner. To assess the efficiency of operation of the biogeochemical cycles, it is important to include both natural and human activities. Often reliable values on use by man are difficult to obtain for a number of reasons, such as lack of international cooperation, and lack of proper bookkeeping and auditing by individual nations. However, a general estimate of the annual world consumption of elements and their compounds is presented.
Stress, radiation and microgravity cause astronauts to experience secondary immunosuppression. Spaceflight conditions enhance bacterial growth and alter antimicrobial susceptibility. Clinostats are used to model microgravity effects at lxg. In controls rotated on the vertical axis, the g-vector acts on cells as in static cultures. Salmonella enterica serovar Typhimurium virulence genes are up-regulated in modelled microgravity (MMG); a MMG regulon has been postulated. We hypothesize that the virulence of P. aeruginosa (PA) may be affected similarly by microgravity, which could be observed in MMG. This study focused on regulation of the ETA protein by PA during growth in MMG. PA103 was grown in an ETA production medium at 37 C. One series of media was inoculated with frozen cultures and grown using horizontal (MMG) or static incubation. Another series inoculated with refrigerated cultures included vertical rotating controls. Analyses included optical density (OD), agar plate counts (PC) on R2A, ETA ELISA, and protein expression by 2-D gel analyses. Growth and ETA results differed depending on inoculum, with minor effects of MMG. Proteomic analysis of 2-D gels indicate differences in protein expression with MMG. Growth and ETA results show that consistent methodology is critical when studying environmental effects. This study provides information on the relationships between environmental changes and virulence regulation, especially for flight experiments, when ground experiments are used to predict potential spaceflight effects.
The NASA Bioculture System is an advanced cell culture closed-loop system containing highly automated flowpaths designed to conduct long term biology experiments on ISS with earth remote controllable medium flow, temperature, gas composition, medium exchange, cell sampling and fixation. This technology was already demonstrated with successful cardiomyocyte and osteocyte cultures experiments onboard the ISS and is now supporting NASA PI science. The Bioculture System, however, can only support 10 cassettes with disposable flowpaths, each containing a single hollow fiber bioreactor with a culture capacity of about 2ml. This constraint not only severely limits the number of investigators that can conduct experiments in space, but also subjects the experiments to limitations in the number of replicates and conditions that can be studied. To address these limitations, we sought a novel design solution to maximize the number of separate bioreactor cultures and volume that can be conducted simultaneously. To this end we designed, prototyped, and are now testing a six-Vitvo 3D Matrix 2ml bioreactor insert that replaces the conventional Bioculture System hollow fiber bioreactor. This design will allow the Bioculture System to support up to 60 different bioreactors and samples at once. Specifically, the novel gas-tight containment housing insert contains six COTS Rigenerand VITVO bioreactors stacked on each side of a heat sink powered by the existing heating element and pair of temperature sensors. Medium will be distributed into each bioreactor's cell-free chamber via its built-in Luer connector, then across the 3D matrix to the cell chamber, dissipating laminar flow and limiting fluid shear stresses that might mechanostimulate cell cultures. Gas (5% CO2 in air) will be supplied directly to the bioreactor gas-tight housing for exchange via the bioreactor flat-surface gas-permeable membranes, eliminating the need for the existing Bioculture System cassette oxygenator. If successfully implemented on ISS, this new multi-bioreactor insert for the Bioculture System has the potential to make real-time cell science experimentation in space more efficient and accessible to more investigators.
Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1 x 10(5) or 22.1 x 10(5) cells per cm2 onto PLGA scaffolds having pore sizes in the range of 150-300 or 500-710 microm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150-300 microm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220+/-40 microm was reached after 56 days in the osteoblast/foam constructs of 150-300 microm pore size initially seeded with a high cell density. For constructs of 500-710 microm pore size, the penetration depth was 190+/-40 microm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150-710 microm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study demonstrates that PLGA foams are suitable substrates for osteoblast growth and differentiated function independent of cell source.
We review the current state of agricultural systems science, focusing in particular on the capabilities and limitations of agricultural systems models. We discuss the state of models relative to five different Use Cases spanning field, farm, landscape, regional, and global spatial scales and engaging questions in past, current, and future time periods. Contributions from multiple disciplines have made major advances relevant to a wide range of agricultural system model applications at various spatial and temporal scales. Although current agricultural systems models have features that are needed for the Use Cases, we found that all of them have limitations and need to be improved. We identified common limitations across all Use Cases, namely 1) a scarcity of data for developing, evaluating, and applying agricultural system models and 2) inadequate knowledge systems that effectively communicate model results to society. We argue that these limitations are greater obstacles to progress than gaps in conceptual theory or available methods for using system models. New initiatives on open data show promise for addressing the data problem, but there also needs to be a cultural change among agricultural researchers to ensure that data for addressing the range of Use Cases are available for future model improvements and applications. We conclude that multiple platforms and multiple models are needed for model applications for different purposes. The Use Cases provide a useful framework for considering capabilities and limitations of existing models and data.
The transition to new technology, innovative ideas, and resistance to change is something that every industry experiences. Recent examples of this shift are changing to using robots in the assembly line construction of automobiles or the increasing use of robotics for medical procedures. Most often this is done with cost-reduction in mind, though ease of use for the customer is also a driver. All industries experience the push to increase efficiency of their systems; National Aeronautics and Space Administration (NASA) and the commercial space industry are no different. NASA space communication services are provided by three separately designed, developed, maintained, and operated communications networks known as the Deep Space Network (DSN), Near Earth Network (NEN) and Space Network (SN). The Space Communications and Navigation (SCaN) Program is pursuing integration of these networks and has performed a variety of architecture trade studies to determine what integration options would be the most effective in achieving a unified user mission support organization, and increase the use of common operational equipment and processes. The integration of multiple, legacy organizations and existing systems has challenges ranging from technical to cultural. The existing networks are the progeny of the very first communication and tracking capabilities implemented by NASA and the Jet Propulsion Laboratory (JPL) more than 50 years ago and have been customized to the needs of their respective user mission base. The technical challenges to integrating the networks are many, though not impossible to overcome. The three distinct networks provide the same types of services, with customizable data rates, bandwidth, frequencies, and so forth. The differences across the networks have occurred in effort to satisfy their user missions' needs. Each new requirement has made the networks more unique and harder to integrate. The cultural challenges, however, have proven to be a significant obstacle for integration. Over the past few decades of use, user missions and network personnel alike have grown accustomed to the processes by which services are provided by the NASA communications and navigation networks. The culture established by each network has created several challenges that need to be overcome in order to effectively integrate the networks. As with any change, there has been resistance, an apprehension to explore automation of existing processes, and a working environment that attempts to indirectly influence change without mandating compliance. Overcoming technical and cultural challenges is essential to successfully integrating the networks and although the challenges are numerous, the integration of the networks promises a more efficient space communications network for NASA and its customers, as well as potential long-term cost savings to the agency. This paper, Challenges of Integrating NASA Legacy Communications Networks, will provide a brief overview of the current NASA space communications networks as well as the an overview of the process implemented while performing the SCaN Trade Studies and an introduction to the requirements driving integration of the SCaN Networks. This paper will describe in detail the challenges experienced, both technical and cultural, while working with NASA space communications network-specific personnel. The paper will also cover lessons learned during the performance of architecture trade studies and provide recommendations for ways to improve the process.
The transition to new technology, innovative ideas, and resistance to change is something that every industry experiences. Recent examples of this shift are changing to using robots in the assembly line construction of automobiles or the increasing use of robotics for medical procedures. Most often this is done with cost-reduction in mind, though ease of use for the customer is also a driver. All industries experience the push to increase efficiency of their systems; National Aeronautics and Space Administration (NASA) and the commercial space industry are no different. NASA space communication services are provided by three separately designed, developed, maintained, and operated communications networks known as the Deep Space Network (DSN), Near Earth Network (NEN) and Space Network (SN). The Space Communications and Navigation (SCaN) Program is pursuing integration of these networks and has performed a variety of architecture trade studies to determine what integration options would be the most effective in achieving a unified user mission support organization, and increase the use of common operational equipment and processes. The integration of multiple, legacy organizations and existing systems has challenges ranging from technical to cultural. The existing networks are the progeny of the very first communication and tracking capabilities implemented by NASA and the Jet Propulsion Laboratory (JPL) more than 50 years ago and have been customized to the needs of their respective user mission base. The technical challenges to integrating the networks are many, though not impossible to overcome. The three distinct networks provide the same types of services, with customizable data rates, bandwidth, frequencies, and so forth. The differences across the networks have occurred in effort to satisfy their user missions' needs. Each new requirement has made the networks more unique and harder to integrate. The cultural challenges, however, have proven to be a significant obstacle for integration. Over the past few decades of use, user missions and network personnel alike have grown accustomed to the processes by which services are provided by the NASA communications and navigation networks. The culture established by each network has created several challenges that need to be overcome in order to effectively integrate the networks. As with any change, there has been resistance, an apprehension to explore automation of existing processes, and a working environment that attempts to indirectly influence change without mandating compliance. Overcoming technical and cultural challenges is essential to successfully integrating the networks and although the challenges are numerous, the integration of the networks promises a more efficient space communications network for NASA and its customers, as well as potential long-term cost savings to the agency. This paper, Challenges of Integrating NASA Legacy Communications Networks, will provide a brief overview of the current NASA space communications networks as well as the an overview of the process implemented while performing the SCaN Trade Studies and an introduction to the requirements driving integration of the SCaN Networks. This paper will describe in detail the challenges experienced, both technical and cultural, while working with NASA space communications network-specific personnel. The paper will also cover lessons learned during the performance of architecture trade studies and provide recommendations for ways to improve the process.
A method and apparatus for screening a sample fluid for bacterial presence are disclosed wherein the fluid sample is mixed with culture media of sufficient quantity to permit bacterial growth in order to obtain a test solution. The concentration of oxygen dissolved in the test solution is then monitored using the potential difference between a reference electrode and a noble metal electrode which are in contact with the test solution. The change in oxygen concentration which occurs during a period of time as indicated by the electrode potential difference is compared with a detection criterion which exceeds the change which would occur absent bacteria.
Muscle cells differentiated in vitro are repetitively stretched and relaxed in order to determine the presence of short- and long-term alterations occurring in glucose uptake and lactate efflux that are similar to the metabolic alterations occurring in stimulated organ-cultured muscle and in vivo skeletal muscle during the active state. It is observed that whereas mechanical stimulation increases these metabolic parameters within 4-6 h of starting activity, unstimulated basal rates in control cultures also increase during this period of time, and by 8 h, their rates have reached or exceeded the rates in continuously stimulated cells. Measurements of these parameters in media of different compositions show that activity-induced long-term alterations in the parameters occur independently of growth factors in serium and embryo extracts.
Cultured human embryonic kidney cells were separated into electrophoretic subpopulations in laboratory experiments and in two separation experiments on the STS-8 (Challenger) Space Shuttle flight using the mid-deck Continuous Flow Electrophoretic Separator (CFES). Populations of cells from each fraction were cultured for the lifetime of the cells, and supernatant medium was withdrawn and replaced at 4-day intervals. Withdrawn medium was frozen at -120 C for subsequent analysis. Enzyme assays, antibodies and gel electrophoresis were used as analytical tools for the detection and quantization of plasminogen activators in these samples. These assays of frozen-culture supernatant fluids confirmed the electrophoretic separation of plasminogen-activator-producing cells from nonproducing cells, the isolation of cells capable of sustained production, and the separation of cells that produce different plasminogen activators from one other.
AIM: As quantitative and spatial analyses of promoter reporter constructs are not easily performed in intact bone, we designed a reporter gene specific to bone, which could be analyzed both visually and quantitatively by using chloramphenicol acetyltransferase (CAT) and a cyan version of green fluorescent protein (GFPcyan), driven by a 2.3-kb fragment of the rat collagen promoter (Col2.3). METHODS: The construct Col2.3CATiresGFPcyan was used for generating transgenic mice. Quantitative measurement of promoter activity was performed by CAT analysis of different tissues derived from transgenic animals; localization was performed by visualized GFP in frozen bone sections. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed for CAT and GFP activity. RESULTS: In mice, CAT activity was detected in the calvaria, long bone, teeth, and tendon, whereas histology showed that GFP expression was limited to osteoblasts and osteocytes. In cell culture, increased activity of CAT correlated with increased differentiation, and GFP activity was restricted to mineralized nodules. CONCLUSION: The concept of a dual reporter allows a simultaneous visual and quantitative analysis of transgene activity in bone.
As NASA lays out its Artemis activities, it expects to set precedents in spaceflight for decades to come. Artemis is a far more ambitious program than even Apollo, aimed at developing a sustained human presence on and around the lunar surface, in preparation for Mars exploration. As a result, considering ethical and social concerns in the discussion and design of Artemis is vital to ensuring the future worlds we create are ones where humanity collectively wants to live. By understanding how to reflect on these issues as NASA makes decisions related to Artemis, NASA can avoid unintended consequences stemming from cultural perceptions of human exploration of the solar system that may harm society. We recognize that a diverse range of groups need to be part of this conversation. There are many different views about the work that should happen in space. We recognize that NASA does not hold sole responsibility for determining the ethical path forward in space, as decisions made by commercial, international, and intergovernmental partners play a major role. Inclusive conversations help inform that work. Others have already argued that social scientists and engineers need to work together with inputs from stakeholders to design the future. To begin to answer this need, NASA’s Office of Technology, Policy, and Strategy (OTPS) hosted an Artemis and Ethics Workshop. This workshop was held in Washington, DC from April 12th-14th, 2023 and brought together invited experts in social science fields and NASA managers to discuss ethical, legal, and social aspects (ELSA) of Artemis and human exploration in general. The goal of the workshop was to discuss issues and build connections that NASA managers can consider across variety of ethical issues surrounding Artemis.
A methodology for growing three-dimensional plant tissue models in a hydrodynamic focusing bioreactor (HFB) has been developed. The methodology is expected to be widely applicable, both on Earth and in outer space, as a means of growing plant cells and aggregates thereof under controlled conditions for diverse purposes, including research on effects of gravitation and other environmental factors upon plant growth and utilization of plant tissue cultures to produce drugs in quantities greater and at costs lower than those of conventional methodologies. The HFB was described in Hydro focus - ing Bioreactor for Three-Dimensional Cell Culture (MSC-22358), NASA Tech Briefs, Vol. 27, No. 3 (March 2003), page 66. To recapitulate: The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear liquid culture environment simultaneously with the herding of suspended cells and tissue assemblies and removal of unwanted air bubbles. The HFB includes a rotating cell-culture vessel with a centrally located sampling port and an internal rotating viscous spinner attached to a rotating base. The vessel and viscous spinner can be made to rotate at the same speed and direction or different speeds and directions to tailor the flow field and the associated hydrodynamic forces in the vessel in order to obtain low-shear suspension of cells and control of the locations of cells and air bubbles. For research and pharmaceutical-production applications, the HFB offers two major benefits: low shear stress, which promotes the assembly of cells into tissue-like three-dimensional constructs; and randomization of gravitational vectors relative to cells, which affects production of medicinal compounds. Presumably, apposition of plant cells in the absence of shear forces promotes cell-cell contacts, cell aggregation, and cell differentiation. Only gentle mixing is necessary for distributing nutrients and oxygen. It has been postulated that inasmuch as cells in the simulated microgravitation of an HFB do not need to maintain the same surface forces as in normal Earth gravitation, they can divert more energy sources to growth and differentiation and, perhaps, to biosynthesis of greater quantities of desired medicinal compounds. Because one can adjust the HFB to vary effective gravitation, one can also test the effects of intermediate levels of gravitation on biosynthesis of various products. The potential utility of this methodology for producing drugs was demonstrated in experiments in which sandalwood and Madagascar periwinkle cells were grown in an HFB. The conditions in the HFB were chosen to induce the cells to form into aggregate cultures that produced anti-cancer indole alkaloids in amounts greater than do comparable numbers of cells of the same species cultured according to previously known methodologies. The observations made in these experiments were interpreted as suggesting that the aggregation of the cells might be responsible for the enhancement of production of alkaloids.
Bone forming cells, osteoblasts, respond to various mechanical forces, including mechanical strain and fluid-induced shear stress. This study examined whether osteoblasts detect changes in gravity as a mechanical force, as assessed by cellular morphology and dimensions of the cytoskeletal network. We used modeling to evaluate how gravity influences cell morphology given theoretical differences in densities between the surrounding medium, cytoplasm, and nucleus. A mechanical model was built based on analysis of axisymmetric shell structures (Fast4 software) to study the effects of 10 times gravity (10G) on cell height. The model indicated 0.02% decrease in overall cell height when the medium was 10% denser than the nucleus or cytoplasm, 5.9 x 10(exp-5)% decrease when the nucleus was 10% denser than the cytoplasm or medium, and 1.3 x 10(exp-5)% decrease when the cell cytoplasm was 10% denser than the nucleus or medium. To experimentally evaluate the influence of gravity, cultured primary fetal rat osteoblasts were grown to near confluence and centrifuged at 10G for 3 hours. Actin, microtubules, and nuclei were fluorescently labeled and analyzed by confocal microscopy to determine overall microtubule and actin network height. Centrifugation led to an apparent reduction in height of both the microtubule (-16%) and the actin (-20%) networks relative to stationary controls. Thus, both modeling and experiments indicate that hypergravity reduces the height of the osteoblast cell layer and their microtubule and actin networks. This combination of modeling and experimental analyses will help us to better understand the mechanical loading of osteoblasts.
To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions by HIMAC at NIRS and NSRL at BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis and neuron survival focusing on the dependencies of the animal strains, SCID, B6, B6C3F1, C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to particle radiation as evaluated by 10% apoptotic criterion. The LET dependency was compared with using SCID and B6 cells exposing to different ions (H, C, Ne, Si, Ar, and Fe). Although no detectable LET dependency was observed in the high LET (55-200 keV/micrometers) and low dose (<0.5 Gy) regions. The survivability profiles of the neurons were different in the mouse strains and ions. In this report, a result of memory and learning function to adult mice after whole-body and brain local irradiation at carbon ion and iron ion.
Representative, reproducible and high-throughput models of human cells and tissues are critical for a meaningful evaluation of host-pathogen interactions and are an essential component of the research developmental pipeline. The most informative infection models - animals, organ explants and human trials - are not suited for extensive evaluation of pathogenesis mechanisms and screening of candidate drugs. At the other extreme, more cost effective and accessible infection models such as conventional cell culture and static co-culture may not capture physiological and three-dimensional aspects of tissue biology that are important in assessing pathogenesis, and effectiveness and cytotoxicity of therapeutics. Our lab has used innovative bioengineering technology to establish biologically meaningful 3-D models of human tissues that recapitulate many aspects of the differentiated structure and function of the parental tissue in vivo, and we have applied these models to study infectious disease. We have established a variety of different 3-D models that are currently being used in infection studies - including small intestine, colon, lung, placenta, bladder, periodontal ligament, and neuronal models. Published work from our lab has shown that our 3-D models respond to infection with bacterial and viral pathogens in ways that reflect the infection process in vivo. By virtue of their physiological relevance, 3-D cell cultures may also hold significant potential as models to provide insight into the neuropathogenesis of HIV infection. Furthermore, the experimental flexibility, reproducibility, cost-efficiency, and high throughput platform afforded by these 3-D models may have important implications for the design and development of drugs with which to effectively treat neurological complications of HIV infection.